A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 μM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 μM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 μM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 μM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.

Introduction

Chromoblastomycosis (CBM) is a subcutaneous neglected mycosis [1]. It is caused by the implantation of one of its etiological agents through a trauma to the skin. Black fungi from the genera Fonsecaea, Cladophialophora, Rhinocladiella, Phialophora, Exophiala, among others, can cause CBM. Since this mycosis is not of compulsory notification in most countries where it occurs, its distribution is based on case reports and case series, with most of them occurring in Latin America, Africa, and Asia. In South America, most of the cases of CBM have been described in Brazil, in Africa most cases come from Madagascar, and China is the leading Asian country on number of cases [2]. In Brazil, the most frequent CBM agents belong to the genus Fonsecaea [3-6]. F. pedrosoi is the predominant species in South America, followed by F. monophora [7]. F. pedrosoi also predominates in humid regions of most countries where CBM is endemic, including Madagascar and China. Cladophialophora carrionii is more frequent in arid areas of these countries [8-10]. The frequency of infections by species belonging to other genera of black fungi varies among different geographic regions [2].

CBM mainly affects agricultural and construction workers, which develop extensive injuries with damage to the affected limb [11]. Most patients often take a long time to seek medical help, therefore their lesions are usually extensive [2]. Although there is no official therapeutic protocol for CBM, itraconazole is the most frequently used drug, followed by terbinafine [2]. In developing countries, including Brazil, treatment based on the use of these drugs is long and expensive, and some patients show recurrence and refractoriness [2,12]. These patients usually require more than one therapeutic method, often including physical methods, such as cryosurgery or laser therapy [13]. The in vitro response of the CBM agents to other antifungal drugs currently used to treat mycotic infections, such as amphotericin B, fluconazole, flucytosine, and micafungin is not satisfactory [14]. A more effective and less time-consuming treatment is clearly required to combat CBM.

The discovery of new pharmacological agents, however, is costly and time-consuming. Moreover, most of compounds in pre-clinical or clinical studies will never be approved for human use [15]. A useful approach to bypass these problems is drug repurposing, where drugs already studied, and sometimes approved, to treat other medical conditions are redirected to target a new disease [16].

In order to identify novel drugs with activity against neglected diseases, the Medicines for Malaria Venture (MMV, Switzerland; http://www.pathogenbox.org/), developed a collection of 400 compounds named Pathogen Box®. This initiative provides the tools for identifying active compounds against neglected pathogens [17,18]. The aim of this study was to identify in vitro antifungal activity within the Pathogen Box® compounds against the CBM agents and to investigate the synergism of these new compounds with drugs currently used to treat CBM.

Materials and methods

Fungal strains and growth conditions

The eight strains used in the study were obtained from the Collection of Pathogenic Fungi (CFP) of Fiocruz, as well as from the American Type Culture Collection (ATCC). Fonsecaea pedrosoi CFP00791 was used throughout the study. This species is one of the major agents of CBM in most regions where this disease is endemic. Cladophialophora carrionii CFP 00910, Phialophora verrucosa CFP 00937, Fonsecaea monophora CFP 00911, Fonsecaea nubica CFP 00912, Rhinocladiela similis CFP 00790, Exophiala jeanselmei var. heteromorpha ATCC 28180, and Exophiala dermatitidis ATCC 28869 were used for minimal inhibitory concentration (MIC) and synergism assays. Strains were maintained on potato dextrose agar (PDA) (Sigma Chemical Corporation, St. Louis, MO, USA). Seven-day-old cultures incubated at 30°C were used in the assays.

The Pathogen Box® compounds

The Pathogen Box® was kindly provided by Medicines for Malaria Venture (MMV, Geneva, Switzerland). It contains 400 different compounds tested for cytotoxicity with values within levels considered acceptable for an initial drug discovery programme [19]. The Pathogen Box® contains compounds with proven activity against neglected pathogens, including those causing tuberculosis, malaria, helminthiasis, cryptosporidiosis, toxoplasmosis, and dengue [20-23]. The compounds were supplied in 96-well microtiter plates containing 10 μl/well of 10 mM compound solutions in dimethylsulfoxide (DMSO). The plates were diluted to a final drug concentration of 1 mM in DMSO (Sigma Chemical Corporation, St. Louis, MO, USA), as recommended by the fabricant, for the drug screening. The first and last columns in each plate were left as blank for negative and positive controls, respectively. All plates were stored at −20°C until their use in the following experiments.

Screening for antifungal activity

For the initial screening, all compounds were tested in 96-well plates (Kasvi Ltda, São José dos Pinhais, PR, Brazil) at a final concentration of 1μM in 100 μl of RPMI 1640 medium, with phenol red, with L-glutamine, and without sodium bicarbonate (Sigma Chemical Corporation), buffered with morpholine propanesulfonic acid (MOPS) (Vetec Química Fina Ltda, Rio de Janeiro, RJ, Brazil) at pH 7.0, and supplemented for a final 2% glucose (Neon Comercial Ltda, São Paulo, SP, Brazil) concentration. The antifungal drugs available as reference compounds in the MMV collection were used as controls of fungal growth inhibition. DMSO concentration in all wells, including those used for fungal control growth, at this point, corresponded to 1%. For preparation of the fungal inoculum, the F. pedrosoi strain CFP 00791 was grown as described above and then its conidia were suspended in sterile distilled water supplemented with 0.1% Tween 20 (Sigma Chemical Corporation) and vortexed, with the suspension turbidity adjusted to the 0.5 McFarland scale. This suspension was further diluted 1:10 and then 100 μl of the fungal inoculum was added to each well containing the compounds, generating a final working inoculum density of 2–5 × 105 CFU/ml and a final DMSO concentration of 0.5% in each well, including controls [21]. Plates were incubated at 35°C for 72–96 hours. The optical density at 530 nm (OD530) was recorded using the Epoch microplate reader (Biotek Instruments Inc, Winooski, VT, USA). The percentage of inhibition of fungal growth (%IG) was calculated according to the formula: %IG = (1- (OD1 / OD2)) × 100, where OD1 = fungal optical density in the presence of the drug; OD2 = optical density of the fungal growth control well without any drug. The %IG data of the 400 compounds was plotted using the Prism 8 software (GraphPad Software, San Diego, CA, USA). Compounds that presented %IG values greater than 60% were selected for further assays.

Minimal inhibitory concentration (MIC) of selected drugs

Assays for MIC determination were performed in sterile polystyrene flat-bottom 96-well microtiter plates using the broth microdilution method according to the EUCAST guidelines [24]. The final concentration of the selected compounds of the Pathogen Box® ranged from 0.015 to 10 μM. All isolates were tested at a final cellular density of 2 to 5 × 105 CFU/ml. Plates were incubated at 35°C for 72–96 h. All experiments were repeated at least two times. The MIC was defined as the lowest drug concentration (μM) that inhibited 100% of fungal growth.

Fungicidal activity

The minimal fungicidal concentration (MFC) was determined by transferring an aliquot of 5 μl of each well without fungal growth of the microdilution plates used for the determination of MIC, as described above, on Sabouraud 2% glucose agar (Sigma Chemical Corporation). The MFC was determined as the lowest drug concentration without fungal growth on the Sabouraud agar after five days of incubation at 35°C. When the MFC:MIC ratio of an agent is 1 or 2, the compound is considered fungicidal against the pathogen. If the ratio is higher than 2, the mode of action is probably fungistatic [25,26].

Evaluation of compounds applicability in CBM treatment

Cytotoxicity data of the compounds was provided by the MMV. The selectivity index (SI) was calculated as follows: SI = CC50 (μM) / MIC (μM), where CC50 is the drug concentration that kills 50% of the cells tested. Compounds with selectivity index lower than 1 were not considered for the synergistic assays. The original use of the compounds and their known side-effects were also verified in the literature, to evaluate their safety for human use. Compounds found to have dangerous side-effects were further excluded from the analysis.

Synergism evaluation

Synergistic activity between the selected compounds and standard antifungal drugs used in the treatment of CBM (itraconazole and terbinafine) was tested by the checkerboard method [27]. Two drugs were loaded into a single 96-well plate, so that in each of the wells there were different concentrations of the compound-antifungal combination. Compound/antifungal dilutions were prepared following the methodology proposed by the EUCAST, starting from a 100-fold concentrated stock compound/antifungal solution according to the MIC determination methodology [24]. Final concentrations of the standard drugs corresponded to 0.06–4 μg/ml (itraconazole) and 0.015–1 μg/ml (terbinafine). Serial two-fold dilutions of the selected Pathogen Box® compounds were performed so that the MIC of that compound was in a central position of the plate (lines 5–7). Fungal inocula and incubation conditions were the same that were described for the previous experiments. The drug interaction was classified according to the fractional inhibitory concentration index (FICI). The FICI was obtained by the formula: FICI ═ (A/MIC (a)) + (B/MIC (b)), where: A = MIC of the drug (a) in combination; MIC (a) = MIC of drug (a) alone; B = MIC of the drug (b) in combination; MIC (b) = MIC of drug (b) alone [28]. The type of interaction between the antifungal agents in combination was classified as the following: synergism if FICI ≤ 0.5; indifference if 0.5 < FICI < 4 and antagonism if FICI ≥ 4 [27,28].

Results

Identification of compounds with antifungal activity against CBM agents

The internal reference drug posaconazole inhibited 99% of the F. pedrosoi growth, validating the assay. Amphotericin B, which is also present in the drug collection, inhibited F. pedrosoi growth by 29%. Among the 400 compounds present in the Pathogen Box®, another eight drugs were found to have antifungal activity against the F. pedrosoi CFP00791 strain (Fig 1). These compounds were identified as difenoconazole (%IG = 98%), bitertanol (%IG = 98%), iodoquinol (%IG = 99%), azoxytrobin (%IG = 95%), MMV688179 (%IG = 73%), MMV021013 (%IG = 62%), trifloxystrobin (%IG = 82%), and auranofin (%IG = 82%). The chemical structure of these compounds is depicted in Fig 2. Their original applications, as described by Medicines for Malaria Venture, are as follows: difenoconazole, bitertanol, MMV688179, and trifloxystrobin have anti-kinetoplastid action, iodoquinol has anti-onchocerciasis action, azoxystrobin has anti-malaria action, MMV021013 has anti-tuberculosis action, and auranofin, a regulated drug for rheumatoid arthritis treatment, is another reference compound present in the collection.

Fig 1

Screening of 400 compounds present in the MMV Pathogen Box® collection against a Fonsecaea pedrosoi strain (CFP00791).

Results are presented as the percent inhibition of growth (%IG) of the fungal strain. The compounds tested are grouped according their original application against some infectious diseases, as described by Medicines for Malaria Venture. The dashed line represents the threshold applied in this study to select compounds with anti-chromoblastomycosis activity. Red symbols represent compounds that were discarded, due to low %IG. Green symbols represent compounds selected for further studies. The blue symbol represents the internal control drug posaconazole.

Fig 2

Chemical structures of the eight compounds with anti-Fonsecaea pedrosoi activity present in the MMV Pathogen Box® collection: (A) MMV688943 (difenoconazole); (B) MMV688942 (bitertanol); (C) MMV688179 (2-[3-chloro-4-[5-[2-chloro-4-(diaminomethylideneamino)phenyl]furan-2-yl]phenyl]guanidine); (D) MMV002817 (iodoquinol); (E) MMV688754 (trifloxystrobin); (F) MMV021013 (N-cyclohexyl-6-cyclopropyl-2-pyridin-2-ylpyrimidin-4-amine); (G) MMV021057 (azoxystrobin); (H) MMV688978 (auranofin).

Efficacy and spectrum of antifungal activity of the selected compounds

Table 1 depicts the MIC and MFC values for each selected compound and the eight CBM agents tested. In brief, iodoquinol (MMV002817) presented the lowest MIC values among the compounds that we tested for antifungal activity. Seven out of the eight strains had MIC values corresponding to 1.25 μM and one MIC was equal to 2.5 μM to this compound. MMV688179 showed the highest MIC values against all species (5 - >10μM). Auranofin (MMV688978) was fungicidal against P. verrucosa and E. dermatitidis, and presented fungistatic activity against the other species. Iodoquinol (MMV002817) and bitertanol (MMV688942) were fungicidal against P. verrucosa and fungistatic against all other species. MMV688179 was fungicidal against P. verrucosa and F. monophora, and fungistatic against the other species. All other compounds had fungistatic activity against the set of isolates tested in this study.

Table 1

Determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) (μM) against chromoblastomycosis agents.

Species	Trifloxystrobin (MMV688754)			Auranofin (MMV688978)			MMV021013			Azoxystrobin (MMV021057)			MMV688179			Difenoconazole (MMV688943)			Iodoquinol (MMV002817)			Bitertanol (MMV688942)
	MIC	MFC	MFC:MIC Ratio	MIC	MFC	MFC:MICRatio	MIC	MFC	MFC:MICRatio	MIC	MFC	MFC:MICRatio	MIC	MFC	MFC:MICRatio	MIC	MFC	MFC: MIC Ratio	MIC	MFC	MFC:MICRatio	MIC	MFC	MFC:MICRatio
C. carrionii	2.5	>10	≥8	1.25	>10	≥16	5	>10	≥4	2.5	>10	≥8	5	>10	≥4	0.625	>10	≥32	1.25	>10	≥16	1.25	10	8
P. verrucosa	2.5	>10	≥8	1.25	2.5	2	5	>10	≥4	0.3125	>10	≥64	5	10	2	2.5	10	4	1.25	1.25	1	5	5	1
E. dermatitidis	10	>10	≥2	2.5	2.5	1	5	>10	≥4	10	>10	≥2	>10	>10	≥1	2.5	10	4	1.25	>10	≥16	10	>10	≥2
E. jeanselmei	0.625	>10	≥32	1.25	10	8	5	>10	≥4	0.3125	>10	≥64	>10	>10	≥1	>10	>10	≥1	1.25	>10	≥16	5	>10	≥4
F. pedrosoi	2.5	>10	≥8	1.25	10	8	2.5	>10	≥8	1.25	10	8	5	>10	≥4	1.25	5	4	1.25	>10	≥16	0.625	10	16
F. monophora	2.5	>10	≥8	1.25	5	4	2.5	>10	≥8	2.5	>10	≥8	5	10	2	2.5	10	4	1.25	10	8	2.5	>10	≥8
F. nubica	0.625	>10	≥32	1.25	>10	≥16	1.25	>10	≥16	2.5	10	4	5	>10	≥4	0.3125	10	32	1.25	>10	≥16	0.3125	10	32
R. similis	2.5	>10	≥8	2.5	>10	≥8	2.5	>10	≥8	0.625	>10	≥32	>10	>10	≥1	2.5	>10	≥8	2.5	>10	≥8	5	>10	≥4

Analysis of synergistic effects

The compound MMV688179 was excluded from further analyses because it showed the highest MIC values (MIC >10 μM) and low selectivity index (Table 2). Trifloxystrobin was found to be toxic to keratinocytes [29], discouraging its use to treat CBM, which affects the skin. From the set of the other agricultural fungicides, we selected bitertanol for synergistic studies, on the basis of its higher selectivity index (Table 2). Therefore, iodoquinol, bitertanol, MMV021013, and auranofin were selected for the determination of FICI in the in vitro combination test with itraconazole and terbinafine. The concentration ranges of the compounds tested corresponded to 0.01–6 μg/ml for auranofin, 0.003–2.5 μg/ml for MMV021013, 0.005–3 μg/ml for bitertanol, and 0.007–4 μg/ml for iodoquinol. The results obtained from the checkerboard method with these four compounds in combination with itraconazole or terbinafine are detailed in Tables 3 and 4, respectively. The auranofin and itraconazole combination produced the single synergistic interaction (FICI < 0.5), against the C. carrionii strain. The combinations of iodoquinol, bitertanol, and MMV021013 with itraconazole produced indifferent interactions (FICI between 0.5 and 4) for all isolates tested. All compounds in combination with terbinafine produced indifferent interactions.

Table 2

Cytotoxicity values, minimal inhibitory concentration (MIC), and Selectivity Index (SI) of selected compounds with activity against the chromoblastomycosis agents.

Species	Trifloxystrobin (MMV688754)			MMV021013			Azoxystrobin (MMV021057)			MMV688179			Difenoconazole (MMV688943)			Iodoquinol (MMV002817)			Bitertanol (MMV688942)
	CC50a	MIC	SI	CC50b	MIC	SI	CC50a	MIC	SI	CC50c	MIC	SI	CC50a	MIC	SI	CC20b	MIC	SI	CC50a	MIC	SI
C. carrionii	>64	2.5	25.6	>50	5	10	>28	2.5	11.2	1	5	0.2	30.9	0.625	49.4	2.5	1.25	2	>64	1.25	51.2
P. verrucosa	>64	2.5	25.6	>50	5	10	>28	0.3125	89.6	1	5	0.2	30.9	2.5	12.3	2.5	1.25	2	>64	5	12.8
E. dermatitidis	>64	10	6.4	>50	5	10	>28	10	2.8	1	>10	0.1	30.9	2.5	12.3	2.5	1.25	2	>64	10	6.4
E. jeanselmei	>64	0.625	102.4	>50	5	10	>28	0.3125	89.6	1	>10	0.1	30.9	>10	3	2.5	1.25	2	>64	5	12.8
F. pedrosoi	>64	2.5	25.6	>50	2.5	20	>28	1.25	22.4	1	5	0.2	30.9	1.25	24.7	2.5	1.25	2	>64	0.625	102.4
F. monophora	>64	2.5	25.6	>50	2.5	20	>28	2.5	11.2	1	5	0.2	30.9	2.5	12.3	2.5	1.25	2	>64	2.5	25.6
F. nubica	>64	0.625	102.4	>50	1.25	40	>28	2.5	11.2	1	5	0.2	30.9	0.3125	98.8	2.5	1.25	2	>64	0.3125	204.8
R. similis	>64	2.5	25.6	>50	2.5	20	>28	0.625	44.8	1	>10	0.1	30.9	2.5	12.3	2.5	2.5	1	>64	5	12.8

Auranofin: not determined (reference compound); CC50 (concentration that killed 50% of the cells) and CC20 (concentration that killed 20% of the cells) values were provided by MMV; Data was obtained with the following cell lines: a MRC5 cells; b HepG2 cells; c PMM cells. SI: selectivity index = CC50 (μM) / MIC (μM). The higher is the ratio obtained, the more selective is the compound against the fungal pathogen.

Table 3

Interaction of selected compounds with itraconazole.

Species	MIC (μg/ml)			FICI AUR/ITZ	MIC (μg/ml)			FICI IOD/ITZ	MIC (μg/ml)			FICI BIT/ITZ	MIC (μg/ml)			FICI MMV021013/ITZ
	AUR	ITZ	AUR/ITZ		IOD	ITZ	IOD/ITZ		BIT	ITZ	BIT/ITZ		MMV021013	ITZ	MMV021013/ITZ
C. carrionii	1.5	0.5	0.375/0.06	0.37*	0.25	0.5	0.007/0.5	1.03	0.375	0.12	0.005/0.06	0.51	2.5	0.5	1.25/0.12	0.74
P. verrucosa	1.5	0.25	0.375/0.12	0.73	0.25	0.25	0.25/0.06	1.24	3.0	0.25	1.5/0.12	0.98	2.5	0.25	1.25/0.06	0.74
E. dermatitidis	1.5	0.25	0.75/0.06	0.74	0.5	0.25	0.25/0.06	0.8	1.5	0.12	0.75/0.06	1	2.5	0.5	0.003/1.0	2
E. jeanselmei	0.75	0.12	0.09/0.06	0.62	0.5	0.06	0.0078/0.06	1	1.5	0.12	0.75/0.06	1	1.25	0.06	0.003/0.06	1
F. pedrosoi	0.75	0.12	0.01/0.12	1	0.25	0.12	0.25/0.015	1.12	0.375	0.12	0.01/0.06	0.52	1.25	0.12	0.625/0.06	1
F. monophora	0.75	0.06	0.01/0.06	1	0.25	0.12	0.125/0.06	1	0.1875	0.12	0.187/0.06	1.5	2.5	0.06	0.003/0.06	1
F. nubica	0.375	0.12	0.01/0.12	1	0.25	0.12	0.125/0.06	1	0.1875	0.12	0.09/0.06	1	0.3125	0.12	0.15/0.06	0.98
R. similis	0.75	0.25	0.75/0.06	1.24	0.5	0.12	0.25/0.06	1	3.0	0.25	0.375/0.25	1.12	2.5	0.25	0.15/0.12	0.54

AUR: auranofin; ITZ: itraconazole; IOD: iodoquinol; BIT: bitertanol; FICI: fractional inhibitory concentration index.

* indicates a synergistic combination.

Table 4

Interaction of selected compounds with terbinafine.

Species	MIC (μg/ml)			FICI AUR/TRB	MIC (μg/ml)			FICI IOD/TRB	MIC (μg/ml)			FICIBIT/TRB	MIC (μg/ml)			FICI MMV021013/TRB
	AUR	TRB	AUR/TRB		IOD	TRB	IOD/TRB		BIT	TRB	BIT/TRB		MMV021013	TRB	MMV021013/TRB
C. carrionii	0.75	0.25	0.75/0.015	1.06	0.50	0.50	0.125/0.25	0.75	0.75	0.12	0.375/0.12	0.98	2.5	0.25	2.5/0.12	1.48
P. verrucosa	0.75	0.06	0.75/0.015	1.25	0.25	0.12	0.125/0.06	1	1.5	0.12	1.5/0.06	1.5	2.5	0.12	1.25/0.015	0.62
E. dermatitidis	0.75	0.25	0.75/0.015	1.06	0.5	0.25	0.25/0.12	0.98	1.5	0.25	1.5/0.12	1.48	5.0	0.25	0.625/0.25	1.12
E. jeanselmei	1.5	0.5	0.75/0.06	0.62	0.50	0.50	0.25/0.03	0.56	1.5	0.25	0.75/0.12	0.98	2.5	0.50	1.25/0.25	1
F. pedrosoi	0.75	0.06	0.375/0.03	1	0.50	0.12	0.25/0.03	0.75	0.375	0.12	0.187/0.06	1	2.5	0.06	0.003/0.06	1
F. monophora	1.5	0.12	0.375/0.12	1	0.50	0.12	0.25/0.03	0.75	0.1875	0.25	0.09/0.06	0.98	2.5	0.12	2.5/0.015	1.12
F. nubica	0.375	0.12	0.005/0.12	1	0.25	0.25	0.25/0.015	1	0.1875	0.12	0.09/0.015	0.62	1.25	0.12	0.625/0.015	0.62
R. similis	0.75	1.0	0.75/0.50	1.50	0.5	1.0	0.5/0.5	1.50	3.0	1.0	1.5/0.5	1	2.5	1.0	2.5/0.50	1.50

AUR: auranofin; TRB: terbinafine; IOD: iodoquinol; BIT: bitertanol; FICI: fractional inhibitory concentration index.

Discussion

CBM is difficult to treat because there is no standardized drug of choice. In addition, relapses are frequent for this mycosis [13]. The taxonomic diversity of the CBM agents adds another obstacle to the development of new drugs against this disease, because most patients are diagnosed exclusively on the basis of the presence of muriform cells in infected tissues [2]. Therefore, general therapeutic decisions are made regardless of the etiology of CBM, which may be deleterious for the patient. In this study, for instance, E. dermatitidis often presented MIC values higher than other species for the studied drugs. In this context, we recommend that repurposing studies for CBM use a large number of etiologic agents of this disease in the antifungal activity experiments.

The compound collection used in this study, the Pathogen Box® library, was previously demonstrated to contain molecules with antifungal activity. Mayer and Kronstad identified five compounds (tolfenpyrad, difenoconazole, bitertanol, posaconazole, and MMV688271) that exhibited antifungal activity against Cryptococcus neoformans and Candida albicans [18]. In our study, antifungal activities were identified for three of these compounds (difenoconazole, posaconazole, and bitertanol). It is noteworthy that tolfenpyrad inhibited F. pedrosoi growth by 49%. This compound is an agricultural insecticide and with well-known poisoning effects [30]. MMV688271, which completely inhibited the growth of C. neoformans and C. albicans [18], showed only 36% inhibition against F. pedrosoi in our screening assay. Vila and Lopez-Ribot evaluated three of the compounds that we investigated (MMV687807, MMV687273, and MMV688768) against C. albicans biofilms [17]. In the present study, these compounds showed 13% (MMV687807), 37% (MMV687273) and 24% (MMV688768) inhibition of the F. pedrosoi growth. Wall et al identified three compounds with anti-C. auris activity in the Pathogen Box® at a 5 μM concentration, including pentamidine, MMV659010, and iodoquinol. In this group, iodoquinol and MMV659010 showed %IG of 86 and 62, respectively, against plant cells. Pentamidine showed 61% inhibition in biofilms of C. auris [31]. In the present study, these compounds presented 34% (MMV659010), 49% (pentamidine), and 99% (iodoquinol) inhibition of the F. pedrosoi growth. These results clearly demonstrate that major fungal pathogens respond differently to the Pathogen Box® compounds (S1 Table).

The MMV Pathogen Box® contains two antifungal drugs as reference compounds. Inhibition of F. pedrosoi growth by posaconazole (99%) was observed, but amphotericin B inhibited less fungal growth (29%). This data can be explained by the high MIC values of amphotericin B (4–16 μg/ml) against this species [14].

In our study, three compounds were selected as promising anti-CBM agents: auranofin, iodoquinol, and MMV021013. Auranofin has been in clinical use as the gold standard for the oral treatment of rheumatoid arthritis since the 1980s [32,33]. The oral bioavailability of auranofin and its reduced side effects offer significant advantages over traditional injectable drugs [32,33]. Several studies were conducted to identify alternative therapeutic applications for auranofin, particularly in the area of infectious diseases. For example, antifungal activity against C. albicans [34] and C. neoformans [34-36] has been described. In this study, low MIC values for this drug against eight different CBM agents were observed. Furthermore, the synergism found in the combination of auranofin with itraconazole against C. carrionii, the main CBM agent in arid and semi-arid areas [37], as well as its fungicidal activity against E. dermatitidis support the use of auranofin as a therapeutic option for CBM. These findings justify future in vivo testing and clinical studies to evaluate auranofin as an antifungal drug.

Iodoquinol (diiodohydroxyquinoline) is a luminal amebicide. Its use is recommended after treatment with a tissue amebicide, such as metronidazole, to eradicate surviving parasites in the colon [38]. This drug has low absorption through the gastrointestinal tract, so only the topical formulation would be applicable against CBM. Burnett and Mitchell demonstrated that the topical formulation of 1% iodoquinol in combination with 2% hydrocortisone acetate had antifungal activity against C. albicans, dermatophytes, and Malassezia furfur [39]. The study by Wall et al. demonstrated the in vitro efficacy of iodoquinol against several Candida species under planktonic conditions. As expected from the initial results with C. auris, iodoquinol was virtually ineffective against preformed biofilms of all species tested. However this compound displayed potent activity against planktonic growth of all strains tested, with IG50 (50% inhibition growth) values generally lower than 1 μg/ml [31]. In the present study, lower MIC values of iodoquinol (100% inhibition) were observed against all studied CBM agents, supporting its use against this group of fungi.

MMV021013 is a 2-pyridyl-4-aminopyrimidine. Antifungal activity has been described for some aminopyrimidines, including4-(2-Fluorophenyl)-6-trifluoromethyl-2-aminopyrimidine, active against Botrytis cinera [40], and 4-(1H-benzo[d]imidazol-2-yl)-6-(p-tolyl)pyrimidin-2-amine, active against Aspergillus flavus, Candida utilis, and Saccharomyces cerevisiae [41]. The Pathogen Box® classifies this compound as a tuberculosis targeted drug. Also, it was found to be active against Leishmania donovani and Trypanosoma cruzi [19]. To the best of our knowledge, no antifungal activity has been described for MMV021013. Some studies have identified molecules with in vitro activity against mycotic and tuberculosis agents [42,43], as well as against protozoa and fungi [44]. These observations suggest that these pathogens may share common antimicrobial targets that may propel drug discovery against a broad range of infectious diseases.

In summary, auranofin was identified in our study as a promising compound to be tested as a CBM therapeutic agent, on the basis of its low MIC values against CBM agents, synergism with itraconazole against a C. carrionii strain, and fungicidal action against P. verrucosa and E. dermatitidis. Iodoquinol has also proved promising for future studies involving CBM agents due to its low MIC values, fungicidal activity against P. verrucosa, and approved topical use in some countries. The 2-pyridyl-4-aminopyrimidine MMV021013 showed good selectivity and a combined activity against mycobacteria, Leishmania, Trypanosoma, and CBM agents. These data suggest the need for further in vitro and in vivo studies with these drugs aiming to develop new therapeutic tools against the CBM agents.

Results are presented as the percent inhibition of growth (%IG) of the fungal strain.

(PDF)

Click here for additional data file.

3 Mar 2020

PONE-D-20-03641

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis

PLOS ONE

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REQUIRED: Both reviewers note confusion related to association between fungistatic vs. fungicidal determinations and recommended clarification of the text related to these studies. Please clarify this text to make sure the studies are easy to understand for a lay reader.

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: In the manuscript “A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis” the authors use the MMV Pathogen Box to identify novel treatments for CBM. An initial screening of 400 compounds against F. pedrosi identified 8 compounds that exhibited antifungal activity. The efficacy of these 8 compounds were then tested against other CBM agents. Additionally, the 8 identified compounds were tested for synergy with itraconazole and terbinafine (two compounds that are commonly used to treat CBM), which ultimately led to the identification of three promising compounds. Throughout the paper, the authors provide good explanations of the methodology, the inclusion of relevant formulas was especially interesting. The analysis provided in this paper would be beneficial for further inquiry into novel CBM therapies. Overall, the paper is well written, and the analyses are explained and presented well.

Major points:

L116: 1% DMSO seems like a high percentage of DMSO, and it could be inhibitory alone to the cells -- was a DMSO control included? If so, this should be included in the text, if not the experiments should be repeated to verify 1% DMSO is not inhibitory or justification for why it was not should be explained.

Table 1: Please provide some clarification on why the MFC value is lower than the MIC value for auranofin against P. verrucosa and for bitertanol against P. verrucosa. And why they are still considered fungistatic? Denoting which drugs were fungicidal would also be helpful in this table, so they are readily identifiable.

L144 -145: “Compounds that presented a MFC value lower than two times the MIC were considered fungicidal.” I am confused by this and need more clarification, especially how this relates to the fungicide determination presented in Table 1. In the results (lines 195-196) the authors state that auranofin is fungicidal against E. dermatidis and iodoquinol is fungicidal against P. verrucosa, but the MFC and MIC values are the same. Perhaps explaining fungicidal determination in terms of MFC to MIC ratio would make it easier for the reader to understand and would be less open to interpretation.

Minor points:

Why was F. pedrosi chosen as the strain to test initially? More background on CBM would be interesting to include in the introduction.

L44-45: Include in the abstract why MMV021013 was thought to be promising. It is included for the other two, but not for the MMV021013.

L65: delete “for”, so it reads “time to seek medical help”

L81: italicize in vitro

L148: Define CC50 in text.

L266 & 267: Keep spelling of amebicide consistent.

Figure 1: I liked that you the compounds were grouped by original application, but it would be nice if the 8 compounds that were studied further were labelled. The markers could also be made smaller just to make it easier to see all the markers.

Figure 2: I am not sure this figure is necessary – perhaps it could be included as a supplementary figure.

Table 2, L478: Unsure what “* Cytotoxicity dada only CC20 Hep G2 cells” means. – Is “dada” supposed to be data?

Reviewer #2: The aims of this study was to identify the in vitro antifungal activity of compounds from the Pathogen Box against Chromoblastomycosis agents. Eight strains from the genera Fonsecaea, Cladophialophora, Rhinocladiella, Phialophora, Exophiala, were tested. Initially 400 compounds were tested against Fonsecaea pedrosoi. Eight compounds were found to have a high inhibition activity against F. pedrosoi were then tested for their MIC and MFC against the 8 CBM agents. After excluding compounds with higher MIC, lower SI and toxicity, 4 final compounds were selected to be tested for a synergistic activity with standards antifungal drugs used to treat CBM. In combination with itraconazole, only one compound (Auranofin) showed synergy against C. carrionii but indifference for other CBM agents. All compounds in combination with terbinafine produced indifferent interactions.

In conclusion, this study identified 3 promising compounds (Auranofin, Iodoquinol and MMV021013) with an anti-CBM activity. This is a well written manuscript and the conclusions are supported by the findings. Indicated below are few question and minor errors that need addressing:

1. Is Auranofin fungistatic against P. verrucosa? Lines 195-196 say that “Auranofin (MMV688978) was fungicidal against the E. dermatitidis strain and 196 fungistatic against the other species”. The definition of a fungicidal drug on line 144 indicate that “Compounds that presented a MFC value lower than two times the MIC were considered fungicidal”. For P. verrucosa MIC=1.25 AND mfc=0.625. Unless I am mistaken I think this MFC value indicate that Auranofin is fungicidal against P. verrucosa too.

2. I will suggest to add what “CC50/CC20” means below Table 2.

3. Minor corrections:

a. Line 81, “in vitro” should be in italic

b. Line 182, add a period before “The chemical structure…”.

c. Line 478, correct “cytotoxicity dada”

**********

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Reviewer #1: Yes: Sophie Altamirano

Reviewer #2: No

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8 Apr 2020

Major points:

Reviewer #1: L116: 1% DMSO seems like a high percentage of DMSO, and it could be inhibitory alone to the cells -- was a DMSO control included? If so, this should be included in the text, if not the experiments should be repeated to verify 1% DMSO is not inhibitory or justification for why it was not should be explained.

Answer: We included a DMSO control on the test board, since we follow the EUCAST protocol that recommends a concentration of 1% DMSO during preparation of the microplates for susceptibility testing. After addition of the fungal inoculum, this concentration drops to 0.5%, which is the final DMSO concentration in the antifungal susceptibility testing. This information was clarified in the new version of the text (lines 122-124 and lines 127-131).

Reviewer #1: Table 1: Please provide some clarification on why the MFC value is lower than the MIC value for auranofin against P. verrucosa and for bitertanol against P. verrucosa. And why they are still considered fungistatic? Denoting which drugs were fungicidal would also be helpful in this table, so they are readily identifiable.

Answer: Thank you for this observation. The MFC lower than the MIC values are really intriguing, therefore we prepared two additional replicates using these compounds against the P. verrucosa isolate. We found MIC values lower or equal to the MFC values. We believe that this one-fold dilution variation on our MFC values were due to intrinsic aspects of the methodology, which has several pipetting and diluting steps that can induce manual errors in the measurements. In fact, several publications only consider two-fold variations on MIC/MFC values as significant. Again, thank you for this observation.

Reviewer #1: L144 -145: “Compounds that presented a MFC value lower than two times the MIC were considered fungicidal.” I am confused by this and need more clarification, especially how this relates to the fungicide determination presented in Table 1. In the results (lines 195-196) the authors state that auranofin is fungicidal against E. dermatidis and iodoquinol is fungicidal against P. verrucosa, but the MFC and MIC values are the same. Perhaps explaining fungicidal determination in terms of MFC to MIC ratio would make it easier for the reader to understand and would be less open to interpretation.

Answer: Thank you for this suggestion. We have changed the definition (lines 152-154) accordingly and also the MFC/MIC ratio was included in table 1 to facilitate the readers’ understanding. Results of fungicidal drugs were also updated in this new version of the manuscript (lines 224-228). Moreover, as mentioned before, we repeated the test twice to confirm the results after receiving the reviewer’s comments.

--------------------

Reviewer #2: Is Auranofin fungistatic against P. verrucosa? Lines 195-196 say that “Auranofin (MMV688978) was fungicidal against the E. dermatitidis strain and 196 fungistatic against the other species”. The definition of a fungicidal drug on line 144 indicate that “Compounds that presented a MFC value lower than two times the MIC were considered fungicidal”. For P. verrucosa MIC=1.25 AND MFC=0.625. Unless I am mistaken I think this MFC value indicate that Auranofin is fungicidal against P. verrucosa too.

Answer: We followed the helpful suggestion of reviewer #1 to address this problem. In the new version of our manuscript we corrected the results related to fungicidal effects of the evaluated drugs against P. verrucosa (lines 224-228).

Reviewer #2: I will suggest to add what “CC50/CC20” means below Table 2.

Answer: Thank you for this suggestion. These definitions were added in table 2 footnotes as requested.

--------------------

Minor points:

Reviewer #1: Why was F. pedrosoi chosen as the strain to test initially? More background on CBM would be interesting to include in the introduction.

Answer: We agree with the reviewer. A description of the species epidemiology on the major endemic areas was included in the first paragraph of introduction (Lines 59-65). In addition, the explanation for using this species in all assays, including the screening, was included in the “fungal strains and culture conditions” section of Materials and methods (Lines 95-96).

Reviewer #1: L44-45: Include in the abstract why MMV021013 was thought to be promising. It is included for the other two, but not for the MMV021013.

Answer #1: We added a sentence about this subject in lines 44-45.

Reviewer #1: L65: delete “for”, so it reads “time to seek medical help”

Answer #1: This was corrected as requested.

Reviewer #1: L81: italicize in vitro

Answer #1: This was corrected as requested.

Reviewer #1: L148: Define CC50 in text.

Answer #1: This definition was included in the new version of the manuscript (lines 157-158)

Reviewer #1: L266 & 267: Keep spelling of amebicide consistent.

Answer: This was corrected as requested.

Reviewer #1: Figure 1: I liked that you the compounds were grouped by original application, but it would be nice if the 8 compounds that were studied further were labelled. The markers could also be made smaller just to make it easier to see all the markers.

Answer: A revised Fig1 file was included in the new version of our manuscript. In this new version of Fig 1, we used colors to differentiate the compounds that were further studied. Also, we used smaller markers and increased the sizes of both x and y axis, to facilitate the visualization of the points. However, there are some classes of compounds (e.g. anti-malaria) with a lot of data points, so it is still difficult to differentiate most of them. But, in our opinion, in this new version of the figure, the compounds with anti-CBM activity are clearly differentiated. We sincerely wish that this new Fig1 file will please the reviewers.

Reviewer #1: Figure 2: I am not sure this figure is necessary – perhaps it could be included as a supplementary figure.

Answer: We preferred to maintain this figure within the manuscript, as usually practiced in similar studies. Moreover, reviewer #2 made a minor comment on our text about the chemical structure of the compounds, so we suppose that the other reviewer also thinks that this figure fits within the main text of our manuscript.

Reviewer #1: Table 2, L478: Unsure what “* Cytotoxicity dada only CC20 Hep G2 cells” means. – Is “dada” supposed to be data?

Answer: This legend was corrected (Line 291). Thanks for this observation.

--------------------

Reviewer #2: Line 81, “in vitro” should be in italic

Answer: This was corrected as requested.

Reviewer #2: Line 182, add a period before “The chemical structure…”.

Answer: This was corrected as requested.

Reviewer #2: Line 478, correct “cytotoxicity dada”

Answer: This was corrected as requested.

We are hopeful that the revised manuscript will be suitable for publication in PLoS One.

Sincerely,

Rowena Alves Coelho.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

23 Apr 2020

PONE-D-20-03641R1

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis

PLOS ONE

Dear Mrs Coelho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The reviewers noted a few very minor edits that were not incorporated into manuscript so it has been returned to correct these errors prior to final acceptance. A detailed response is not required, just acknowledgement that the corrections have been made to the submitted manuscript.

==============================

We would appreciate receiving your revised manuscript by Jun 07 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: N/A

Reviewer #2: N/A

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All of my comments were addressed, and I think the authors made all of the necessary revisions.

Minor

L154: Change fungicide to fungicidal

Reviewer #2: The authors has addressed most of comments on the previous version of the manuscript. However, modifications added on lines 225-226 were not added in the final summary (lines 381-382). I will suggest adding in the final summary that auranofin is also fungicidal against P. verrucosa.

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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24 Apr 2020

After the last review the changes suggested by reviewers # 1 and # 2 have been corrected. Thanks for this observation.

Reviewer #1: All of my comments were addressed, and I think the authors made all of the necessary revisions.

Minor

L154: Change fungicide to fungicidal

Answer: This was corrected as requested.

Reviewer #2: The authors has addressed most of comments on the previous version of the manuscript. However, modifications added on lines 225-226 were not added in the final summary (lines 381-382). I will suggest adding in the final summary that auranofin is also fungicidal against P. verrucosa.

Answer: This was corrected as requested.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

28 Apr 2020

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis

PONE-D-20-03641R2

Dear Dr. Coelho,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Reviewers' comments:

30 Apr 2020

PONE-D-20-03641R2

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis

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